A root cap-localized NAC transcription factor controls root halotropic response to salt stress in Arabidopsis

Plants are capable of altering root growth direction to curtail exposure to a saline environment (termed halotropism). The root cap that surrounds root tip meristematic stem cells plays crucial roles in perceiving and responding to environmental stimuli. However, how the root cap mediates root halotropism remains undetermined. Here, we identified a root cap-localized NAC transcription factor, SOMBRERO (SMB), that is required for root halotropism. Its effect on root halotropism is attributable to the establishment of asymmetric auxin distribution in the lateral root cap (LRC) rather than to the alteration of cellular sodium equilibrium or amyloplast statoliths. Furthermore, SMB is essential for basal expression of the auxin influx carrier gene AUX1 in LRC and for auxin redistribution in a spatiotemporally-regulated manner, thereby leading to directional bending of roots away from higher salinity. Our findings uncover an SMB-AUX1-auxin module linking the role of the root cap to the activation of root halotropism.

Values are means ± SD.In b and c, n indicates the number of independent seedlings, and alphabets denote significant differences (P < 0.05, one-way ANOVA by Tukey's test).

Supplementary Fig. 2 .a
Comparison of sensitivity between wild-type and SMB knockout mutants to different salt gradients.Related to Fig. 1.Halotropic root response of Col-0 and seedlings that were transferred to split-agar medium containing different concentrations of NaCl for 1 day (a) and 3 days (c).White arrows indicate the direction of NaCl diffusion; white dotted lines represent the initial location of the root tip when the salt gradient was created; orange dotted line represents mock-mock or mock-salt boundary; orange arrows represent the occurrence of halotropic root bending; while red arrows indicate the absence of halotropic root bending.Scale bar, 1 cm.Halotropic root curvature was quantified at 1 day (b) and 3 days (d).e Quantification of primary root elongation in Col-0 and seedlings that were transferred to split-agar medium containing different concentrations of NaCl for 1 day and 3 days.Values are means ± SD.In b, d and e, n indicates the number of independent seedlings, and alphabets denote significant differences (P < 0.05, two-way ANOVA by Tukey's test).Supplementary Fig. 3.The effects of SMB on root halotropic bending and root elongation.Related to Fig. 1.Halotropic root curvatures of Col-0, , and its complemented line seedlings that were transferred to split-agar medium with or without 250 m NaCl for 3 days.The angles were grouped on the wheel diagram as percentage of root bending.Black and blue bars represent the ock and NaCl treatments, respectively.b Quantification of primary root elongation in Col-0, , and its complemented line seedlings that were transferred to split-agar medium with or without 250 m NaCl for 1 day and 3 days.Values are means ± SD.In a and b, n indicates the number of independent seedlings, and alphabets indicate significant differences (P < 0.05, two-way ANOVA by Tukey's test).
Halotropic root response of Col-0 and 5 R seedlings that were transferred to split-agar medium containing 150 m and 250 m NaCl in the absence or presence of indicated DEX concentrations for 3 days (a).White arrows indicate the direction of NaCl diffusion; white dotted lines represent the initial location of the root tip when the salt gradient was created; orange dotted line represents mock-salt boundary.Yellow arrows note enhanced halotropic root response of 5 R roots upon DEX treatments, compared with Col-0 under corresponding treatments (orange arrows).Scale bar, 1 cm.Halotropic root curvature was quantified and shown in (b).c Quantification of primary root elongation in Col-0 and 5 R seedlings that were transferred to split-agar medium containing different concentrations of NaCl in the absence or presence of indicated DEX concentrations for 3 are means ± SD.In b and c, n indicates the number of independent seedlings, and alphabets indicate significant differences (P < 0.05, two-way ANOVA by Tukey's test).Supplementary Fig. 5.The effects of SMB overexpression on halotropic root response for 1d.Related to Fig. 1. a, b Halotropic root response of Col-0 and 5 R seedlings that were transferred to split-agar medium containing varying NaCl concentrations in the absence or presence of indicated DEX concentrations for 1 day (a).White arrows indicate the direction of NaCl diffusion; white dotted lines represent the initial location of the root tip when the salt gradient orange dotted line represents mock-mock or mock-salt boundary.Yellow arrows note enhanced halotropic root response of 5 R roots upon DEX treatments, compared with Col-0 under corresponding treatments (orange arrows).Scale bar, 1 cm.Halotropic root curvature was quantified and shown in (b).c Quantification of primary root elongation in Col-0 and 5 R seedlings that were transferred to split-agar medium containing different concentrations of NaCl in the absence or presence of indicated DEX concentrations for 1 day.Values are means ± SD.In b and c, n indicates the number of independent seedlings, and alphabets indicate significant differences (P < 0.05, two-way ANOVA by Tukey's test).Supplementary Fig. 6.Effects of SMB on endogenous sodium level, plant fresh weight, and salt-tolerance associated gene expression under salt stress.Related to Fig. 1. a, b Na + content and Na + /K + radio of Col-0 and seedlings that were transferred to homogeneous NaCl (100 m )-containing 1/2 S plates for 3 and 10 days (n 3 biological repeats).c Quantification of the net Na + influx in the root tips of Col-0 and seedlings treated with 100 m NaCl (n 3 biological repeats).d Quantification of fresh weight of Col-0 and seedlings that were transferred to a split-agar medium with or without 250-m NaCl for 9 days.e Quantification of fresh weight of Col-0 and seedlings that were transferred to medium with or without 100 m NaCl for 7 days.f qPCR analysis of gene expression in the roots of Col-0 and seedlings that were transferred to a split-agar medium with or without 250 m NaCl for 6 hours (n 3 biological repeats).Values are means ± SD.In d and e, n indicates the number of independent seedlings.Statistical analysis was performed with two-tailed Student's t test (P < 0.05; ns, not significant).Analysis of pSMB:nls-GFP and pSMB:SMB-GFP/smb-3 expression in root tips at the early stage of halo-stimulation.Related to Fig. 1. a qRT-PCR analysis of transcript in the root of Col-0 and seedlings that were transferred to split-agar medium with or without 250 m NaCl for 6 hours.Values are means ± SD of three biological replicates.Statistical analysis was performed with two-tailed twotailed Student's t test (P < 0.05; ns, not significant).b-e Expression patterns (b and d) and quantifications (c and e) of pS B:nls-3xVENUS and S B-G P in the root tips of respective Col-0 and seedlings that were transferred to split-agar medium with or without 250 m NaCl over 4 hours.The fluorescence images were overlapped with bright-field images.Similar results were obtained in three independent experiments.Scale bar, 100 μm.The fluorescence intensities of pS B:nls-3xVENUS (b) and S B-G P (d) in the LRC and epidermis located distal (left) or proximal (right) to mock/salt gradient were quantified.Each point represents an individual data point.Values are means ± SD.In c and e, n indicates the seedlings.Statistical analysis was performed with two-tailed Student's t test (P < 0.05; ns, not significant).Supplementary Fig. 8.The effect of SMB on gravitropic root growth under different concentrations of NaCl.Related to Fig. 2. a A schematic diagram defining VGI (See aterial and methods).Ly: vertical primary root length (root depth; the orange line); L: total root length (the pink curved line).VGI Ly/L.b Quantification of VGI of Col-0 and seedlings grown in vertical plates after 5 days germination.Values are means ± SD (n 39 seedlings); statistical analysis was performed with two-tailed Student's t test (***P < 0.001).c-e Gravitropic root response of Col-0 and seedlings that were transferred to medium with varying concentrations of NaCl and rotated 90 degrees for 2 days gravi-stimulation.The picture was taken 2 days after treatments VGI .White arrows represent the position of the root tips when treatment was initiated, and red arrows indicate the position of the root tips after 2 days gravi-stimulation.Scale bar,1 cm.Gravitropic root curvature was quantified and shown in (d).0º equals vertical angle.Primary root elongation was quantified and shown in (e).Values are means ± SD.In b, d and e, n indicates the number of independent seedlings, and alphabets denote significant differences (P < 0.05, two-way ANOVA by Tukey's test).Supplementary Fig. 9.Staining of amyloplasts in the columella cells of Col-0 and smb-3 under salt stress.Related to Fig. 2. a-c Amyloplasts staining and quantification of the root apexes of Col-0 and seedlings that were transferred to medium containing different concentrations of NaCl at indicated time points (a).Similar results were obtained in three independent experiments.Scale bar,100 μm.Amyloplasts area of the root apex was quantified via Lugol's staining (b) and mPS-PI (c).
Halotropic root responses of Col-0, , 4 , adg , , 4 and adg seedlings that were transferred to split-agar medium with or without 250 m NaCl (a).White arrows indicate the direction of NaCl diffusion; white dotted lines represent the initial location of the root tip when the salt gradient was created; orange dotted line represents mock-mock or mock-salt boundary; orange arrows represent the occurrence of halotropic root bending, while red arrows indicate the absence of halotropic root bending.Yellow arrows indicate accelerated root halotropic bending in the indicated genotypes, relative to that of Col-0 (orange arrows); and red arrows indicate compromised root halotropic response.Scale bar, 1 cm.Halotropic root curvature was quantified and shown in (b).c Primary root elongation of indicated genotypes were transferred to split-agar medium with or without 250 m NaCl for 1 day and 3 days.Values are means ± SD.In b and c, n indicates the number of independent seedlings, and alphabets denote significant differences (P < 0.05, two-way ANOVA by Tukey's test).Supplementary Fig. 12. Gravitropic of starch synthesis mutants ss4-3 and adg1-1.Related to Fig. 2. a-c Gravitropic root response of Col-0 and 4 , and adg after 1 day gravi-stimulation.Petri dishes with germinated seedlings were rotated 90 degrees for gravistimulation.White arrows represent the initial position of the root tips when gravistimulation was started, and while red arrows indicate the position of the root tips after 1 day gravi-stimulation (a).Scale bar,1 cm.Gravitropic root curvature was quantified and shown in (b).Violin plot showing the distribution.Lines represent the upper, median and lower quartile data distribution.Primary root elongation was quantified and shown in (c).Values are means ± SD.In b and c, n indicates the number of independent seedlings.Statistical analysis was performed with twotailed Student's t test (*P < 0.05; ns, not significant).Quantification of lateral root cap cell number and columella cell layer in Col-0 and smb-3 upon halo-stimulation.a, b Confocal image of the root apexes of Col-0 and seedlings that were transferred to split-agar medium with or without 250 m NaCl for 6 hours.The roots of seedlings were cleaned by Clearsee (a) or stained with PI (b) before imaging.Orange arrow points to the columella cell layer, and each white triangle points to a lateral root cap (LRC) cell.The yellow asterisk represents epidermis cells, and white arrows indicate the direction of NaCl diffusion.Similar results were obtained in three independent experiments.Scale bar,100 μm.c Quantification of LRC number and columella cell layers of Col-0 and seedlings that were transferred to split-agar medium with or without 250 m NaCl for 6 hours.Values are means ± SD; n indicates the number of independent seedlings.Statistical analysis was performed with two-tailed Student's t test (P < 0.05; ns, not significant).The effects of auxin antagonists on gravitropic and halotropic root responses of Col-0 and smb-3.Related to Fig. 3. a, b Gravitropic root response of Col-0 and seedlings after 1 day gravi-stimulation in the absence or presence of 10 μ PEO-IAA, 10 μ Yucasin or 1 μ L-Kyn.Petri dishes with rotated 90 degrees for gravi-stimulation.White arrows represent the position of the root tips when gravi-stimulation was initiated, and red arrows indicate the position of the root tips after one day gravi-stimulation (a).Scale bar,1 cm.Gravitropic root curvature was quantified and shown in (b).Violin plot showing the distribution.Lines represent the upper, median and lower quartile data distribution.Statistical analysis was performed with two-tailed Student's t test (***P < 0.001).c-e Halotropic root response of Col-0 and seedlings that were transferred to split-agar medium with or without 250 m NaCl in the absence or presence of 10 μ PEO-IAA, 10 μ Yucasin or 1 μ L-Kyn for 1 day.White arrows indicate the direction of NaCl diffusion; white dotted lines represent the initial location of the root tip when the salt gradient was created; orange dotted line represents mock-mock or mock-salt boundary.Yellow arrows note enhanced halotropic root response of indicated genotypes upon compounds treatments, relative to that of Col-0 (orange arrows); and red arrows indicate the absence of halotropic root bending (c).Scale bar, 1 cm.Halotropic root curvature (d) and primary root elongation (e) were quantified and shown.Values are means ± SD.Alphabets denote significant differences (P < 0.05, two-way ANOVA by Tukey's test).f, g Confocal images (f) and quantification (g) of DR5rev:3xVENUS-N7 signal intensity in the root tips of indicated genotypes during 6 hours of halo-stimulation.White arrows indicate the direction of NaCl diffusion (f).Similar results were obtained in three independent experiments.Scale bar,100 μm.DR5rev:3xVENUS-N7 signal intensity at the both sides of LRC and epidermal cells was quantified (g).Values are means ± SD.In b, d, e and g, n indicates the number of independent seedlings.Statistical analysis was performed with two-tailed Student's t test (*P < 0.05; ns, not significant).Auxin accumulation in root cap is sufficient to affect auxin accumulation in root tip and halotropic root response.Related to Fig. 3. a, b Confocal image of the root apexes of Col-0 and iaaH seedlings that were transferred to medium with or without 1 μ IA treatment for 6 hours (a).Orange arrow points c cell layer, and each red triangle points to a lateral root cap cell.The yellow asterisk represents epidermis cells (a).Similar results were obtained in three independent experiments.Scale bar, 50 μm.LRC number of Col-0 and iaaH was measured and shown in (b).Values are means ± SD.Statistical analysis was performed with two-tailed Student's t test (P < 0.05; ns, not significant).c-f Halotropic root response of Col-0 after 24 hours of halo-stimulation in the presence or absence of 1 μ IA .White arrows indicate the direction of NaCl diffusion; white dotted lines represent the initial location of the root tip when the salt gradient was created; orange dotted line represents mock-salt boundary; orange arrows represent the occurrence of halotropic root bending (c).Scale bar, 100 μm.Halotropic root curvature (d) and primary root elongation (e) was quantified and shown.Violin plot showing the distribution.Lines represent the upper, median and lower quartile data distribution.Values are means ± SD.Statistical analysis was performed with two-tailed Student's t test (P < 0.05; ns, not significant).f Primary root elongation of iaaH seedlings after 1 day and 3 days of halo-stimulation in the absence or presence of 1μ IA .Values are means ± SD.Statistical analysis was performed with two-tailed Student's t test (***P < 0.001; ns, not significant).g-i Expression patterns of R5 (g) and (h) in the root tips of Col-0 and iaaH treated with or without 1 μ IA for 2 days.Similar results were obtained in three independent experiments.Scale bar, 100 μm.DII:VENUS signal intensities in LRC and epidermis cell were quantified and shown in (i).Values are means ± SD.In b, d, e, f and i, n indicates the number of independent seedlings.Statistical analysis was performed with two-tailed Student's t test (***P < 0.001; ns, not significant).Supplementary Fig. 18.Effects of NAA on auxin accumulation and halotropic root response of Col-0 and smb-3.Related to Fig. 3. a, b Confocal images of DR5rev:3xVENUS-N7 signal in LRC and epidermal cells of Col-0 and after 6 hours of halo-stimulation in the presence of 0.3 µ NAA.White arrows indicate the direction of NaCl diffusion (a).Similar results were obtained in three independent experiments.Scale bar, 100 μm.Radio of DR5 signal intensity at distal salt (Ds)/proximal salt (Ps) sides of LRC and epidermal cells over 6 hours was quantified and shown in (b).Values are means ± SD.Statistical analysis was performed with two-tailed Student's t test (P < 0.05; ns, not significant).c-e Halotropic root response of Col-0 and seedlings that were transferred to split-agar medium without NaCl in the presence of 0.3 µ NAA for 1 day and 3 days (c).White dotted lines represent the initial location of the root tip when the salt gradient was created; orange dotted line represents mock-mock boundary; red arrows indicate the absence of halotropic root bending.Scale bar, 100 μm.Halotropic root curvature (d) and primary root elongation (e) was quantified and shown.Violin plot showing the distribution.Lines represent the upper, median and lower quartile data distribution.Statistical analysis was performed with two-tailed Student's t test (P < 0.05; ns, not significant).Values are means ± SD.In b, d and e, n indicates the number of independent seedlings, and alphabets denote significant differences (P < 0.05, two-way ANOVA by Tukey's test).
of halo-stimulation (b).White arrows indicate the direction of NaCl diffusion; white dotted lines represent the initial location of the root tip when the salt gradient was created; orange dotted line represents mock-mock or mock-salt boundary.Yellow arrows indicate accelerated root halotropic bending in the indicated genotypes, relative to that of Col-0 (orange arrows); and red arrows indicate compromised root halotropic response.Scale bar, 1 cm.Primary root elongation at 1 day and 3 days of halo-stimulation was quantified and shown in (c).Values are means ± SD. n indicates the number of independent seedlings, and alphabets indicate significant differences (P < 0.05, two-way ANOVA by Tukey's test).Supplementary Fig. 21.Halotropic root response of aux1-21 and aux1-21 smb-3 upon halo-stimulation.Related to Fig. 4. a-e Halotropic root response of au and au seedlings that were transferred to split-agar medium containing different concentrations of NaCl for 1 day (a) and 3 days (d).White arrows indicate the direction of NaCl diffusion; white dotted lines represent the initial location of root tip when the salt gradient was created; orange dotted line represents mock-